Method of judging viral infection

ABSTRACT

A method for diagnosing a viral infection without complicating bacterial infection, which comprises determining a C-reactive protein and an MxA protein in a biological sample; a kit for judging viral infection without complicating bacterial infection, which comprises a reagent for determining a C-reactive protein and a reagent for determining an MxA protein in a biological sample.

TECHNICAL FIELD

The present invention relates to a method for diagnosing viralinfections and a kit for the judgment.

BACKGROUND ART

Since the discovery of penicillin in 1928, it has become possible totreat considerable bacterial infections by antibiotics which have beendeveloped and improved one after another. However, this fact does notmean that all infections including bacterial infections can be treated.Many patients suffering from infections still need to be treated andhence it has been desired to develop a diagnostic method for selecting atreatment method. However, there exists no clear and rapid method fordistinction between bacterial infections and viral infections inclinical practice at the present stage.

Until now, for the distinction of bacterial infections and otherinfections, symptoms in patients, the number of peripheral leukocytesand C-reactive protein values are employed. The C-reactive protein isone of acute phase response substances and is known as a marker fordiagnosis and progress observation of various infections. The C-reactiveprotein value in blood of a healthy adult is 0.06 mg/dL. The case wherethe C-reactive protein value in blood is from 1 to 10 mg/dL is diagnosedto be a disorder of medium-grade increase and the case where the valueis 10 mg/dL or higher is diagnosed to be a disorder of high-gradeincrease. When the diagnosis is a disorder of medium-grade increase,chronic rheumatism, angiitis, rheumatic fever, malignant tumor, cardiacinfarction, and external injury are suspected in addition to bacterialinfections. Moreover, when the diagnosis is a disorder of high-gradeincrease, serious bacterial infections and active rheumatism aresuspected [Clinical Test Method Summary (Rinsho Kensa Ho Teiyo),published by Kanehara Shuppan, 500 (1998)].

As mentioned above, it is difficult to diagnose bacterial infectionsperfectly with the C-reactive protein value in blood which is employedfor diagnosis of bacterial infections.

Recently, MxA proteins have attracted attention as specific markers forviral infections. MxA proteins are induced in lymphocytes by type Iinterferons (IFN-α and IFN-β) which are increased at viral infection,but are not at all affected by the other inflammatory cytokines such asIL-1, IL-6 and TNF-α of which productions are increased in bacterialinfections and chronic inflammatory diseases [Eur J Immunol, 20, 2015(1990)]. Therefore, the expression of an MxA protein in lymphocytes isconsidered to be utilized for detection of all viral infections and apossibility of its clinical application has been reported [Pediatr Res,41, 647 (1997)].

In medical sites, there is a tendency that antibiotics are used forpatients when they are suspected to suffer from bacterial infections[JAMA, 273, 213 (1995)]. Such use of antibiotics has resulted in aserious problem of occurrence of multidrug-resistant bacteria such asmethicillin-resistant Staphylococcus aureus (MRSA) which are resistantto various antibiotics.

Apporopriate use of antibiotics is expected to be able to contribute notonly to prevention of the appearance of multidrug-resistant bacteria butalso to suppression of increase of medical costs which is currentlyrecognized as a problem [Pedicatrics, 108, 1 (2001)]. Therefore, it isimportant to clearly distinguish between viral infections and bacterialinfections, but the method to distinguish them has not been known untilnow.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a method for diagnosinga viral infection without complicating bacterial infection and a reagentused for the diagnosis.

The present invention relates to a method for diagnosing a viralinfection without complicating bacterial infection, which comprisesdetermining a C-reactive protein and an MxA protein in a biologicalsample.

Moreover, the present invention relates to a kit for diagnosing a viralinfection without complicating bacterial infection, which comprises areagent for determining a C-reactive protein and a reagent fordetermining an MxA protein in a biological sample.

In order to diagnose a viral infection without complicating bacterialinfection, the C-reactive protein and the MxA protein are measured.

The method for determining a C-reactive protein is not particularlylimited, so long as it is a method for determining a C-reactive protein.Examples include an immunological determining method using an antibodyagainst a C-reactive protein. As the immunological determining method,any method is included, so long as it uses an antigen-antibody reaction,such as an immunoassay, an immunoblotting method, an agglutinationreaction, a complement-fixation reaction, a hemolytic reaction, aprecipitation reaction, a gold colloid method, a chromatography methodor an immunostaining method, and an immunoassay is preferred.

The immunoassay is a method for detecting or determining an antibody orantigen by using variously labeled antigens or antibodies. The methodincludes a radioimmunoassay (RIA), an enzyme immunoassay (EIA or ELISA),a fluorescent immunoassay (FIA), a luminescent immunoassay, aphysicochemical assay (TIA, LAPIA, PCIA), a flow cytometry and the like,depending on the method of labeling the antigens or antibodies. As themethod for determining a C-reactive protein, a physicochemical assay ispreferred.

Specifically, the assay is carried out by binding a C-reactive proteinas an antigen to an antibody or antiserum which specifically binds tothe C-reactive protein to thereby form an agglutinate and by detectingthe agglutinate. The other physicochemical assays include methods fordetermination using a capillary method, a one-dimensionalimmunodiffusion method, an immunonephelometry, a lateximmunonephelometry or the like [Clinical Test Method Summary (RinshoKensa Ho Teiyo), published by Kanehara Shuppan, 499 (1998)].

For example, in the latex immunonephelometry, when an antigen-antibodyreaction is induced on a polystyrene latex having a particle size ofabout 0.1 to 1 μm immobilized with an antibody or antigen by using acorresponding antigen or antibody, scattered light increases whiletransmitted light decreases in the reaction mixture. The C-reactiveprotein can be determined by measuring the change as absorbance orintegrating sphere turbidity. Specific reagents for determining aC-reactive protein include Determiner T CRP and Extel CRP manufacturedby Kyowa Medex Co., Ltd., N-Assay TIA-CRP-H Kit manufactured by NittoBoseki Co., Ltd., and the like.

The method for determining an MxA protein is not particularly limited,so long as it is a method for determining an MxA protein. Examplesinclude an immunological determining method using an antibody against anMxA protein. As the immunological determining method, any method can beused, so long as it uses an antigen-antibody reaction, such as animmunoassay, an immunoblotting method, an agglutination reaction, acomplement-fixation reaction, a hemolytic reaction, a precipitationreaction, a gold colloid method, a chromatography method or animmunostaining method. As the method for determining an MxA protein, animmunoassay is preferred.

The immunoassay is a method for detecting or determining an antibody orantigen by using variously labeled antigens or antibodies. The methodincludes a radioimmunoassay (RIA), an enzyme immunoassay, (EIA orELISA), a fluorescent immunoassay (FIA), a luminescent immunoassay, aphysicochemical assay (TIA, LAPIA, PCIA), a flow cytometry and the like,depending on the method of labeling the antigen or antibody, and a flowcytometry is preferred.

As a fluorescent label used in a flow cytometry, any known fluorescentlabels (Akira Kawaoi, Fluorescent Antibody Method (Keiko Koutai Ho),published by Soft Science Inc.) can be employed. For example, FITC, RITCand the like can be used.

Specifically, an antibody or antiserum specifically binding to an MxAprotein is allowed to react with peripheral lymphocytes subjected toparaffin fixing, the antibody specifically bound to an MxA proteinexpressed in the lymphocytes is then allowed to react with afluorescence-labeled appropriate secondary antibody, and difference offluorescent intensity is detected by a flow cytometry [WO96/05230,Allergy, 56, 895 (2001)].

The kit for diagnosing a viral infection without complicating bacterialinfection according to the present invention comprises a reagent fordetermining a C-reactive protein and a reagent for determining an MxAprotein. The kit of the present invention may be a combination ofindividual independent kits of the reagent for determining a C-reactiveprotein and the reagent for determining an MxA protein as well as a kitcontaining the reagent for determining a C-reactive protein and thereagent for determining an MxA protein in one. Moreover, in the kitcomprising the reagent for determining a C-reactive protein and thereagent for determining an MxA protein in one, common reagents of therespective reagents can be commonly used. The common reagents include adiluting solution for a biological sample, an antibody-immobilized solidphase, a reaction buffer, a washing liquid, a labeled secondary antibodyor antibody fragment thereof, a reagent for detecting a label, and thelike. The kit of the present invention can be combined with aninstrument suitable for the measurement to form a further kit.

Even when the constitution or form is different, any kit is included inthe kits of the present invention, so long as it contains substancesessentially the same as individual constitutional elements of thereagents for determining a C-reactive protein or the reagents fordetermining an MxA protein or essentially the same as a part thereof.

The reagent for determining a C-reactive protein includes an antibodyreactive to a C-reactive protein, and also include, if necessary, adiluting solution for a biological sample, an antibody-immobilized solidphase, a reaction buffer, a washing solution, a labeled secondaryantibody or antibody fragment thereof, a reagent for detecting a label,a standard substance of a C-reactive protein, and the like.

The antibody reactive to a C-reactive protein is not particularlylimited, so long as it is an antibody reactive to a C-reactive protein,and examples include commercially available anti-human C-reactiveprotein polyclonal antibodies of goat, rabbit, rat and the like, and amurine anti-human C-reactive protein monoclonal antibody (manufacturedby SIGMA).

The diluting solution for a biological sample includes aqueous solutionscontaining proteins such as BSA or casein in addition to a surfactant, abuffer and the like.

The antibody-immobilized solid phase includes a solid phase wherein anantibody or antibody fragment is immobilized on a material obtainable byshaping various polymer materials so as to conform to the use. The shapeincludes a tube, beads, a plate, fine particles such as latex, a stickand the like. The material includes polymer materials such aspolystyrene, polycarbonate, polyvinyltoluene, polypropylene,polyethylene, polyvinyl chloride, nylons, polymethacrylates, gelatin,agarose, cellulose and polyethylene terephthalate, glass, ceramics,metals and the like. As methods for immobilizing an antibody, knownmethods are employed, e.g., physical methods, chemical methods, andcombination thereof. Examples include a solid phase obtainable byhydrophobically immobilizing an antibody or the like on a 96-wellmicrotiter plate for immunoassay made of polystyrene.

The reaction buffer may be any buffer, so long as the antibody on theantibody-immobilized solid phase is capable of binding to the antigen ina biological sample, and examples include phosphate buffers, Good'sbuffer and the like. Moreover, if necessary, a surfactant, a proteinsuch as BSA or casein, an antiseptic, a stabilizer, a reactionaccelerator and the like can be added.

The washing solution includes a phosphate buffer saline (hereinafterreferred to as “PBS”) containing 0.05% Tween 20 and the like. Moreover,if necessary, a surfactant, a protein such as BSA or casein, anantiseptic, a stabilizer or the like can be added.

The labeled secondary antibody or antibody fragment thereof includes anantibody or antibody fragment obtained by binding a labeling enzyme suchas horseradish peroxidase, bovine small intestine alkaline phosphataseor β-galactosidase to the antibody or antibody fragment. To thesecondary antibody or antibody fragment thereof, if necessary, a buffer,a protein such as BSA or casein, an antiseptic or the like can be added.

As the reagent for detecting the label, according to the above labelingenzyme, for example, a substrate for absorption photometry, such astetramethylbenzidine or o-phenylenediamine, a fluorescent substrate suchas hydroxyphenyl hydroxyphenylpropionate or hydroxyphenylacetatic acid,or a luminescent substrate such as luminol can be used for horseradishperoxidase, and a substrate for absorption photometry such as4-nitrophenyl phosphate, a fluorescent substrate such as4-methylumbelliferyl phosphate, or the like can be used for alkalinephosphatase.

The standard substance includes C-reactive proteins obtained byrecombinant DNA techniques or obtained from a biological sample, cellsin which a C-reactive protein is expressed, and the like.

The reagent for determining an MxA protein includes an antibody reactiveto an MxA protein, and also includes, if necessary, a diluting solutionfor a biological sample, an antibody-immobilized solid phase, a reactionbuffer, a washing solution, a labeled secondary antibody or antibodyfragment thereof, a reagent for detecting a label, a standard substanceof an MxA protein, and the like.

The antibody reactive to an MxA protein is not particularly limited, solong as it is an antibody reactive to an MxA protein, and examplesinclude an anti-MxA protein antibody KM1135 produced by a hybridoma FERMBP-4731 (WO96/05230).

The diluting liquid for a biological sample includes aqueous solutionscontaining proteins such as BSA or casein, in addition to a surfactant,a buffer and the like.

The antibody-immobilized solid phase includes a solid phase wherein anantibody or antibody fragment is immobilized to a material obtainable byshaping various polymer materials so as to conform to the use. The shapeincludes a tube, beads, a plate, fine particles such as latex, a stick,and the like. The material includes polymer materials such aspolystyrene, polycarbonate, polyvinyltoluene, polypropylene,polyethylene, polyvinyl chloride, nylons, polymethacrylates, gelatin,agarose, cellulose and polyethylene terephthalate, glass, ceramics,metals, and the like. As methods for immobilizing an antibody, knownmethods are employed, e.g., physical methods, chemical methods, andcombination thereof. Examples include a solid phase obtainable byhydrophobically immobilizing an antibody or the like on a 96-wellmicrotiter plate for immunoassay made of polystyrene.

The reaction buffer to be incorporated in the reagent for determining anMxA protein may be any buffer, so long as the antibody of theantibody-immobilized solid phase is capable of binding to the antigen ina biological sample, and examples include a phosphate buffer, a Good'sbuffer and the like. Moreover, if necessary, a surfactant, a proteinssuch as BSA or casein, an antiseptic, a stabilizer, a reactionaccelerator or the like can be added.

The washing solution may be any washing solution, so long as it iscapable of washing those which do not participate in theantigen-antibody reaction for determining an MxA protein, and examplesinclude fetal bovine serum and PBS containing 0.1% azide and the like.Moreover, if necessary, a buffer, a surfactant, a protein such as BAS orcasein, an antiseptic, a stabilizer or the like can be added.

The labeled secondary antibody or antibody fragment thereof includes anantibody or antibody fragment obtainable by binding a labeling enzymesuch as horseradish peroxidase, bovine small intestine alkalinephosphatase or β-galactosidase to the antibody or antibody fragment. Tothe secondary antibody or antibody fragment thereof, if necessary, abuffer, a protein such as BSA or casein, an antiseptic or the like canbe added.

As the reagent for detecting the label, according to the above labelingenzyme, for example, a substrate for absorption photometry, such astetramethylbenzidine or o-phenylenediamine, a fluorescent substrate suchas hydroxyphenyl hydroxyphenylpropionate or hydroxyphenylacetic acid, ora luminescent substrate such as luminol can be used for horseradishperoxidase, and a substrate for absorption photometry such as4-nitrophenyl phosphate, a fluorescent substrate such as4-methylumbelliferyl phosphate, and the like can be used for alkalinephosphatase.

The standard substance includes MxA proteins obtained by recombinant DNAtechniques or obtained from a biological sample, cells in which an MxAprotein is expressed, and the like.

As the biological sample used in the present invention, for example,blood can be used.

In the present invention, the C-reactive protein and the MxA protein aredetermined and based on each observed value, the presence or absence ofthe expression of the C-reactive protein and the MxA protein is judged,so that a viral infection without complicating bacterial infection isdiagnosed.

In this case, the case where the C-reactive protein is negative and theMxA protein is positive is diagnosed to be a viral infection withoutcomplicating bacterial infection. On the other hand, the case where theC-reactive protein is positive and the MxA protein is negative isdiagnosed to be a bacterial infection without complicating viralinfection. The case where the C-reactive protein is positive and the MxAprotein is positive is diagnosed to be a viral infection with abacterial infection. The case where the C-reactive protein is negativeand the MxA protein is negative is diagnosed to be unknown.

In the present invention, the criteria for diagnosis as positive ornegative on each observed value of the C-reactive protein and the MxAprotein can be suitably decided depending on a determining method. Forexample, when the C-reactive protein in blood is measured by animmunoassay, the diagnosis is carried out by considering a value of lessthan 1.0 mg/dl to be negative and a value of 1.0 mg/dl or more to bepositive. Moreover, when the MxA protein is measured by an immunoassay,an average value and standard deviation on healthy persons in whom noviral infection is observed are obtained, and the diagnosis is carriedout by considering a value of the average value+3SD or more to bepositive and a value of less than the above value to be negative.

Specific examples are shown below.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1

The following measurements were carried out by using peripheral bloodsamples of 74 persons who were suspected to suffer from any ofinfections and from whom blood samples were collected for various assay.

The collected peripheral blood samples were immediately injected intoserum-submitting spits tubes for determining a C-reactive protein valuein blood and for the other general assay, and into a spits tubecontaining heparin Na for determining an MxA protein, and were treatedwithin the day.

The C-reactive protein value in blood was measured by using N-ASSAYTIA-CRP-H kit manufactured by Nitto Boseki Co., Ltd. according to theattached protocol. The observed C-reactive protein value in blood wasjudged by using a value of 1.0 mg/dl as a standard value and byconsidering patients having a value of less than 1.0 mg/dl to benegative and patients having a value of 1.0 mg/dl or more to bepositive.

The expression of the MxA protein in lymphocytes was analyzed asfollows.

After 200 μl of peripheral blood collected with heparin was fixed with4% paraformaldehyde, erythrocytes were destroyed and lymphocyticmembrane was made permeable with a lysis buffer containing 0.1% TritonX-100. Then, staining was carried out with the anti-MxA protein antibodyKM1135 produced by a hybridoma cell FERM BP-4731 or a primary antibodyof an anti-murine IgG1 antibody as a control antibody. After washing,color was developed by using a secondary antibody, and analysis wascarried out with a flow cytometer, and the difference of thefluorescence intensity and that of the control antibody is considered tobe an MxA protein value. Cord blood in normal childbirth was used forobtaining a standard value, and for convenience' sake, a fluorescenceintensity of its average value+3SD or more was used as a standard value.It was diagnosed that patients showing the average value+3SD or lesswere negative for expression of an MxA protein in lymphocytes andpatients showing the average value+3SD or more was positive forexpression of an MxA protein in lymphocytes.

With regard to a fever, patients having a maximum body temperature onthe blood-collected day of 37.5° C. or higher was judged to have a feverand patients having a maximum body temperature of 37.5° C. or lower wasjudged to have no fever.

Table 1 shows results obtained by classifying these 74 patients underjudgment based on a fever, judgment based on the C-reactive proteinvalue in blood, and judgment based on the expression of the MxA proteinin lymphocytes. TABLE 1 Patients having a Patients having no fever feverTotal number of patients 48 26 C-reactive protein value in 31 6 blood(positive) (64.6%) (23.1%) Expression of MxA protein 33 16 inlymphocytes (positive) (68.8%) (61.5%)

The ratios of the patients who were positive in the diagnosis based onthe C-reactive protein value in blood were 64.6% in the case that theyhad a fever and 23.1% in the case where they had no fever. On the otherhand, the ratios of the patients who were positive based on theexpression of the MxA protein in lymphocytes were 68.8% in the casewhere they had a fever and 61.5% in the case where they had no fever.Therefore, the positive diagnosis based on the C-reactive protein valuein blood and the positive diagnosis based on the expression of the MxAprotein in lymphocytes were found to be attributable to independentjudgment criteria and hence a possibility that much information wasobtained by combining both of them was shown.

Actually, when 74 patients were analyzed by combining the diagnosisbased on the C-reactive protein value in blood and the diagnosis basedon the expression of the MxA protein in lymphocytes, there were somecases where causes of diseases could be identified in patients whosediseases had been diagnosed as simple bacterial infections or whosecauses of the diseases had been unknown.

There were 31 patients who had a fever and were diagnosed to be positivebased on the C-reactive protein value in blood, and their diseases hadhitherto been diagnosed as bacterial infections. When the diagnosisbased on the expression of the MxA protein in lymphocytes was combinedfor the 31 patients, patients who were positive in the expression of theMxA protein in lymphocytes were found to be 19 patients. Among the 19patients, 5 cases of asthma attack and 5 cases of possible viraladenoiditis were included and thus existence of patients who were alsoconsidered to suffer from viral infections was revealed. This fact showsthat the diagnosis method of the present invention is superior to theconventional diagnosis method wherein the case that a fever is observedand the based on the C-reactive protein value in blood is positive isdiagnosed to be a bacterial infection.

Additionally, even when a fever was observed, the causes of 17 patientswho were diagnosed to be negative based on the C-reactive protein valuein blood had been hitherto diagnosed to unknown. When the diagnosisbased on the expression of the MxA protein in lymphocytes was combinedfor the 17 patients, the patients who were diagnosed to be positivebased on the expression of the MxA protein in lymphocytes were found tobe 14 patients. Actually, among the 14 patients, 4 cases of asthmaattack, 2 cases of viral gastroenteritis (suspected), and 2 cases ofviral encephalitis (suspected) were included.

Patients who had no fever and were negative in the diagnosis based onthe C-reactive protein value in blood and hence who were diagnosed notto suffer from bacterial infection were found to be 20 patients. Whenthe diagnosis based on the expression of the MxA protein in lymphocyteswas combined for the 20 patients, patients who were positive in thediagnosis based on the expression of the MxA protein in lymphocytes wasfound to be 14 patients.

Actually, among the 14 patients, viral infection was confirmed in 5patients and it was confirmed that 4 patients suffered from RS virusinfection and 1 person suffered from congenital cytomegalovirusinfection.

Therefore, even in the cases that the judgment based on the C-reactiveprotein value in blood was negative and thus the causes of diseases wereunknown in the past, it has been found that viral infections can easilybe diagnosed and therapeutic strategy can be rapidly decided bycombining the diagnosis based on the expression of the MxA protein inlymphocytes.

INDUSTRIAL APPLICABILITY

According to the present invention, a method for diagnosing a viralinfection without complicating bacterial infection and a kit used forthe method are provided.

A viral infection without complicating bacterial infection can bediagnosed by analyzing a C-reactive protein and an MxA proteinsimultaneously by using the method for diagnosing a viral infectionwithout complicating bacterial infection and the kit used for the methodaccording to the present invention.

1. A method for diagnosing a viral infection without complicatingbacterial infection, which comprises determining a C-reactive proteinand an MxA protein in a biological sample, and judging the present orabsent of the expression of the C-reactive protein and the MxA proteinbased on determined value.
 2. A kit for diagnosing a viral infectionwithout complicating bacterial infection, which comprises a reagent fordetermining a C-reactive protein and a reagent for determining an MxAprotein in a biological sample.